Project #1: Elimination of mossy cell NMDA receptors results in impaired neurogenesis. Previously, we subjected mossy cell NR1-KO mice and their control littermates to a battery of behavioral tasks including fear conditioning, a task that involves associating a particular context with an aversive stimulus. We found that mutants older than 15 weeks old, but not younger mutants, show reduced immediate-foot-shock freezing compared to their control littermates when the foot shock was weak, suggesting impaired sensorimotor integration of weak stimuli. This year, we explored the mechanisms underlying these impaired behaviors, and found a profound impairment in dentate neurogenesis. Interestingly, a decrease in the expression of neurogenesis markers, including Doublecortin (DCX) was detected after postnatal 15 weeks, 5-6 weeks after completion of NR1 knockout in the mossy cells, but temporally correlated with the emergence of the behavioral deficits. The manuscript is in preparation. Project #2: Elimination of mossy cells impairs contextual pattern separation but does not induce epileptogenesis. Earlier, we generated a conditional transgenic mouse line in which diphtheria toxin receptor was selectively expressed in mossy cells using the Cre/loxP system. Within one week after diphtheria toxin injection, we observed 80% loss of mossy cells throughout the longitudinal axis. We found no obvious or sustained epilepsy-like discharges in the hippocampus as measured by in vivo local field potential recordings. Interestingly, no mossy fiber sprouting was detected by Timm staining. These results suggested that, in contrast to previous reports showing that lesions of the entire hilar region induce massive mossy fiber sprouting and epilepsy, selective in vivo elimination of mossy cells does not trigger behavioral epilepsy or mossy fiber sprouting. This year, we found that dentate granule cells in the DT-treated mutants became hyperexcitable to afferent stimulation in in vitro slice preparation, and during this hyperexcitable state deficits in contextual pattern separation were detected. We also evaluated the immediate-early gene (IEG) expression in response to kainic acid (KA) injection under the assumption that an excitatory stimulus would cause more granule cells to discharge and activate IEG expression in mutants compared to controls. KA injection evoked Zif268 expression in more granule cells in mutants than in controls. We also examined the KA-induced seizure intensity. The cumulative seizure score of mutants for the hour following KA injection was significantly higher than controls. Together, these results all suggested an increase in granule cell excitability following mossy cell ablation. In summary, we concluded that mossy cell loss in vivo renders the granule cells hyperexcitable. Contrary to the predicted epileptogenesis implicit in the dormant basket cell hypothesis, however, it was insufficient to trigger the mossy fiber sprouting and epileptic discharges. Perhaps, in addition to the loss of mossy cells, neurodegeneration of other limbic areas, such as entorhinal cortex, is necessary to induce medial temporal lobe epilepsy. These findings provide new insights into the mechanisms of epileptogenesis in the limbic cortex. The manuscript is under revision for publication in Neuron.